Agricultural soils are the primary anthropogenic source of atmospheric nitrous oxide (N2OO), contributing to global warming and depletion of stratospheric ozone. Biochar addition has shown potential to lower soil N2OO emission, with the mechanisms remaining unclear. We incubated eucalypt biochar (550 °C) - 0, 1 and 5% (w/w) in Ferralsol at 3 water regimes (12, 39 and 54% WFPS) - in a soil column, following gamma irradiation. After N2OO was injected at the base of the soil column, in the 0% biochar control 100% of expected injected N2OO was released into headspace, declining to 67% in the 5% amendment. In a 100% biochar column at 6% WFPS, only 16% of the expected N2OO was observed. X-ray photoelectron spectroscopy identified changes in surface functional groups suggesting interactions between N2OO and the biochar surfaces. We have shown increases in -O-C = N /pyridine pyrrole/NH3, suggesting reactions between N2OO and the carbon (C) matrix upon exposure to N2OO. With increasing rates of biochar application, higher pH adjusted redox potentials were observed at the lower water contents. Evidence suggests that biochar has taken part in redox reactions reducing N2OO to dinitrogen (N2), in addition to adsorption of N2OO.